Neurons communicate with each other via chemical signals across synapses, a fundamental process that underlies all brain function. Even small aberrations during synaptic development may lead to neurological disease. A growing body of biochemical and genetic data has found that synapse formation occurs during development as the pre-synaptic neuron attaches to the post-synaptic cell via specific adhesion proteins, ensuring that there is close enough proximity between the two cells to permit synapse maturation. Changes in the native expression of specific adhesion proteins have been linked to autism-spectrum disorders in mice, and some individuals with autism spectrum disorders have genetic mutations in their DNA that codes for expression of these specific adhesion proteins. While it is known that these adhesion proteins control the interaction of the pre-synaptic and post-synaptic neurons, a number of questions remain about the mechanism of neuron-neuron interaction, including kinetics, the sequence of binding events, and reversibility or permanence of the interactions. In order to follow individual events over the course of synapse formations, it is necessary to use an imaging probe that is bright and photostable enough to last minutes to hours under the intensity required for single molecule tracking. The probe will also need to be small enough so as not to perturb the interaction of the adhesions proteins. Although a variety of imaging probes are available for single protein tracking in live cells, none are have the stability over the timescale of synapse development. The proposed research details the development of bioimaging probes using upconverting nanoparticles that are bright, non-blinking and photostable over hours-properties that will permit us to follow the events of synapse development in real time. Combinatorial methods will be employed to synthesize nanoparticles that are small (sub-10 nm diameter) and show bright, visible luminescence after near-infrared (NIR) excitation. After synthesis, the nanoparticles with be functionalized to interact with specific neuronal adhesion proteins. Subsequent experiments wil use the developed nanoparticles in live-cell imaging of single proteins in neurons to determine more information about how the specific proteins are involved in synapse maturation and to uncover details as to how perturbations in this process can lead to neurological diseases such as autism. PUBLIC HEALTH RELEVANCE: The described research efforts are focused on understanding how specific neuronal proteins affect synapse formation and maturation. These proteins are of interest because individuals with genetic mutations that cause changes in these proteins have been diagnosed with autism spectrum disorders. A more detailed knowledge of the role of these proteins is critical for the development of diagnostics and treatment for autism.